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Conduct Disorder (CD) is an impairing psychiatric disorder of childhood and adolescence characterized by aggressive and dissocial behavior. Environmental factors such as maternal smoking during pregnancy, socio-economic status, trauma, or early life stress are associated with CD. Although the number of females with CD is rising in Western societies, CD is under-researched in female cohorts. We aimed at exploring the epigenetic signature of females with CD and its relation to psychosocial and environmental risk factors. We performed HpaII sensitive genome-wide methylation sequencing of 49 CD girls and 50 matched typically developing controls and linear regression models to identify differentially methylated CpG loci (tags) and regions. Significant tags and regions were mapped to the respective genes and tested for enrichment in pathways and brain developmental processes. Finally, epigenetic signatures were tested as mediators for CD-associated risk factors. We identified a 12% increased methylation 5’ of the neurite modulator SLITRK5 ( FDR = 0.0046) in cases within a glucocorticoid receptor binding site. Functionally, methylation positively correlated with gene expression in lymphoblastoid cell lines. At systems-level, genes (uncorr. P < 0.01) were associated with development of neurons, neurite outgrowth or neuronal developmental processes. At gene expression level, the associated gene-networks are activated perinatally and during early childhood in neocortical regions, thalamus and striatum, and expressed in amygdala and hippocampus. Specifically, the epigenetic signatures of the gene network activated in the thalamus during early childhood correlated with the effect of parental education on CD status possibly mediating its protective effect. The differential methylation patterns identified in females with CD are likely to affect genes that are expressed in brain regions previously indicated in CD. We provide suggestive evidence that protective effects are likely mediated by epigenetic mechanisms impairing specific brain developmental networks and therefore exerting a long-term effect on neural functions in CD. Our results are exploratory and thus, further replication is needed.

Background Altered neuronal development is discussed as the underlying pathogenic mechanism of autism spectrum disorders (ASD). Copy number variations of 16p11.2 have recurrently been identified in individuals with ASD. Of the 29 genes within this region, quinolinate phosphoribosyltransferase ( QPRT ) showed the strongest regulation during neuronal differentiation of SH-SY5Y neuroblastoma cells. We hypothesized a causal relation between this tryptophan metabolism-related enzyme and neuronal differentiation. We thus analyzed the effect of QPRT on the differentiation of SH-SY5Y and specifically focused on neuronal morphology, metabolites of the tryptophan pathway, and the neurodevelopmental transcriptome. Methods The gene dosage-dependent change of QPRT expression following Chr16p11.2 deletion was investigated in a lymphoblastoid cell line (LCL) of a deletion carrier and compared to his non-carrier parents. Expression of QPRT was tested for correlation with neuromorphology in SH-SY5Y cells. QPRT function was inhibited in SH-SY5Y neuroblastoma cells using (i) siRNA knockdown (KD), (ii) chemical mimicking of loss of QPRT, and (iii) complete CRISPR/Cas9-mediated knock out (KO). QPRT - KD cells underwent morphological analysis. Chemically inhibited and QPRT-KO cells were characterized using viability assays. Additionally, QPRT-KO cells underwent metabolite and whole transcriptome analyses. Genes differentially expressed upon KO of QPRT were tested for enrichment in biological processes and co-regulated gene-networks of the human brain. Results QPRT expression was reduced in the LCL of the deletion carrier and significantly correlated with the neuritic complexity of SH-SY5Y. The reduction of QPRT altered neuronal morphology of differentiated SH-SY5Y cells. Chemical inhibition as well as complete KO of the gene were lethal upon induction of neuronal differentiation, but not proliferation. The QPRT-associated tryptophan pathway was not affected by KO. At the transcriptome level, genes linked to neurodevelopmental processes and synaptic structures were affected. Differentially regulated genes were enriched for ASD candidates, and co-regulated gene networks were implicated in the development of the dorsolateral prefrontal cortex, the hippocampus, and the amygdala. Conclusions In this study, QPRT was causally related to in vitro neuronal differentiation of SH-SY5Y cells and affected the regulation of genes and gene networks previously implicated in ASD. Thus, our data suggest that QPRT may play an important role in the pathogenesis of ASD in Chr16p11.2 deletion carriers.

Objective The phenotypic and genotypic spectrum of adult patients with epilepsy and intellectual disability (ID) is less clear than in children. We investigated an adult patient cohort to further elucidate this and inform the genetic testing approach. Methods Fifty‐two adult patients (30 male, 22 female) with epilepsy, at least mild ID and no known genetic or acquired cause were included and phenotyped. Variants identified through exome sequencing were evaluated using ACMG criteria. Identified variants were compared with commercially available gene panels. Cluster analysis of two features, age at seizure onset and age at ascertainment of cognitive deficits, was performed. Results Median age was 27 years (range 20‐57 years) with median seizure onset at 3 years and median ascertainment of cognitive deficits at 1 year. Likely pathogenic/pathogenic variants were identified in 16/52 patients (31%) including 14 (27%) single nucleotide variants and 2 (4%) copy number variants. Simulated yield of commercial gene panels varied between 13% in small (≤144 genes) and 27% in large panels (≥1478 genes). Cluster analysis (optimal number 3 clusters) identified a cluster with early seizure onset and early developmental delay (developmental and epileptic encephalopathy, n = 26), a cluster with early developmental delay but late seizure onset (ID with epilepsy, n = 16) and a third cluster with late ascertainment of cognitive deficits and variable seizure onset (n = 7). The smaller gene panels particularly missed the genes identified in the cluster with early ascertainment of cognitive deficits and later onset of epilepsy (0/4) as opposed to the cluster with developmental and epileptic encephalopathy (7/10). Significance Our data indicates that adult patients with epilepsy and ID represent a heterogeneous cohort that includes grown‐up patients with DEE but also patients with primary ID and later onset of epilepsy. To maximize diagnostic yield in this cohort either large gene panels or exome sequencing should be used.

Mutations affecting mTOR or RAS signaling underlie defined syndromes (the so-called mTORopathies and RASopathies) with high risk for Autism Spectrum Disorder (ASD). These syndromes show a broad variety of somatic phenotypes including cancers, skin abnormalities, heart disease and facial dysmorphisms. Less well studied are the neuropsychiatric symptoms such as ASD. Here, we assess the relevance of these signalopathies in ASD reviewing genetic, human cell model, rodent studies and clinical trials. We conclude that signalopathies have an increased liability for ASD and that, in particular, ASD individuals with dysmorphic features and intellectual disability (ID) have a higher chance for disruptive mutations in RAS- and mTOR-related genes. Studies on rodent and human cell models confirm aberrant neuronal development as the underlying pathology. Human studies further suggest that multiple hits are necessary to induce the respective phenotypes. Recent clinical trials do only report improvements for comorbid conditions such as epilepsy or cancer but not for behavioral aspects. Animal models show that treatment during early development can rescue behavioral phenotypes. Taken together, we suggest investigating the differential roles of mTOR and RAS signaling in both human and rodent models, and to test drug treatment both during and after neuronal development in the available model systems.

Hintergrund:Autismus-Spektrum-Störungen (ASS) umfassen eine Reihe von genetisch komplexen Störungen mit hoher Erblichkeit. Als zugrundeliegender Pathomechanismus von ASS werden unter anderem Veränderungen der neuronalen Entwicklung diskutiert. ASS ist definiert durch Einschränkungen in der sozialen Interaktion und Kommunikation sowie durch repetitives und stereotypes Verhalten. Genkopiepolymorphismen (englisch „copy number variations“/CNVs), also Deletionen oder Duplikationen einer chromosomalen Region, wurden wiederholt in Probanden mit ASS identifiziert. Dabei gelten Deletionen im Allgemeinen als verheerender, da sich die Reduktion der Gen-Dosis meist stärker auf den Phänotypen auswirkt als eine Hochregulierung (Chang et al., 2015). Die in ASS mit am häufigsten von CNVs betroffene Region liegt auf Chromosom 16p11.2 und umspannt mit einer Größe von ~600kb insgesamt 29 Gene (Woodbury-Smith and Scherer, 2018). Einige dieser Gene wurden bereits funktionell charakterisiert. Zum Beispiel konnte das Gen KCTD13 in einer Zebrafischstudie als verantwortliches Gen für Veränderungen der Kopfgröße identifiziert werden; ein Phänotyp welcher auch bei humanen Trägern eines 16p11.2 CNVs beobachtet wurde (Golzio et al., 2012; Steinman et al., 2016). Während Träger einer 16p11.2 Deletion sowie das Zebrafisch-Modell mit reduzierter KCTD13-Gendosis häufig eine Makrozephalie entwickeln, wird in Duplikationsträgern bzw. Zebrafischen mit hochreguliertem KCTD13 von einer Mikrozephalie berichtet. Dennoch können die bisherigen Einzelgenstudien nicht alle Aspekte erklären, die durch CNVs der Region 16p11.2 hervorgerufen werden. Ziel dieser Studie war es daher, ein weiteres neuronal assoziiertes Kandidatengen dieser Region zu identifizieren und im Anschluss funktionell im Kontext der neuronalen Differenzierung zu charakterisieren.Methoden:Zunächst wurde die SH-SY5Y Neuroblastomzelllinie auf ihre Eigenschaften als Modell für die neuronale in-vitro Differenzierung untersucht. Während der 11-tägigen Differenzierung mittels Retinsäure (RA) und dem Zytokin „brain derived neurotrophic factor“ (BDNF) wurden die Zellen mittels Microarray auf Transkriptomebene und mittels Sholl-Analyse (Ristanović et al., 2006) auf morphologischer Ebene charakterisiert. Drei komplementäre statistische Methoden wurden verwendet, um differenziell regulierte Gene zu identifizieren (Chiocchetti et al., 2016). Mittels „weighted gene co-expression network analysis“ (WGCNA) wurden Gene zu ko-regulierten Modulen zusammengefasst. Zusätzlich wurden auf RNA- (Real-time reverse Transkriptase/RT-PCR) und Protein-Ebene (Western Blot) Marker der Zellteilung bzw. der neuronalen Differenzierung analysiert. Die Expression der 16p11.2 Gene wurde in Hinblick auf die Expressionshöhe sowie deren Veränderung über die Zeit miteinander verglichen. Mittels real-time PCR wurde eine vermutliche de novo Deletion der Region 16p11.2 in der DNA eines Probanden mit ASS untersucht und gegen die DNA seiner gesunden Eltern verglichen. Als erster Schritt der funktionellen Validierung wurde über real-time RT-PCR die Gendosis-abhängige Expression von QPRT in lymphoblastoiden Zelllinien (englisch „lymphoblastoid cell lines“/LCLs) des Deletions-Trägers sowie dessen Eltern analysiert. In SH-SY5Y Zellen wurde die Expression des Kandidatengens QPRT auf Korrelation mit neuromorphologischen Parametern getestet. Die Funktion von QPRT wurde in SH-SY5Y auf drei Ebenen gehemmt: (i) mittels knock down (KD) durch siRNA, (ii) durch chemische Inhibition mit Phthalsäure und (iii) über gezielten knock out (KO) durch CRISPR/Cas9 gesteuerte Geneditierung. Die Morphologie von differenzierenden KD-Zellen wurde mittels Sholl-Analyse untersucht und gegen Kontrollzellen verglichen. Zellen mit chemischer Inhibition oder KO des Kandidatengens wurden über Viabilitäts-Assays charakterisiert. Stimulationen durch Quinolinsäure (QUIN), dem Substrat von QPRT, wurden in WildtypZellen durchgeführt und über Viabilitäts-Assays gemessen, um die durch den KO bedingte vermutete Anreicherung von QUIN zu imitieren. In KO-Zellen wurde zudem versucht die Exzitotoxizität von QUIN durch Antagonisten und Inhibitoren der QUIN-Zielstrukturen zu hemmen, um so den KO-Effekt zu kompensieren. Zusätzlich wurden in den KO-Zellen die Metaboliten des QPRT-assoziierten Tryptophanstoffwechsels mittels Gaschromatographie/Massenspektrometrie sowie Ultrahochleistungsflüssigkeitschromatographie untersucht.

Autism spectrum disorders (ASD) are highly heritable and are characterized by deficits in social communication and restricted and repetitive behaviors. Twin studies on phenotypic subdomains suggest a differing underlying genetic etiology. Studying genetic variation explaining phenotypic variance will help to identify specific underlying pathomechanisms. We investigated the effect of common variation on ASD subdomains in two cohorts including >2500 individuals. Based on the Autism Diagnostic Interview-Revised (ADI-R), we identified and confirmed six subdomains with a SNP-based genetic heritability h 2 SNP  = 0.2–0.4. The subdomains nonverbal communication (NVC), social interaction (SI), and peer interaction (PI) shared genetic risk factors, while the subdomains of repetitive sensory-motor behavior (RB) and restricted interests (RI) were genetically independent of each other. The polygenic risk score (PRS) for ASD as categorical diagnosis explained 2.3–3.3% of the variance of SI, joint attention (JA), and PI, 4.5% for RI, 1.2% of RB, but only 0.7% of NVC. We report eight genome-wide significant hits—partially replicating previous findings—and 292 known and novel candidate genes. The underlying biological mechanisms were related to neuronal transmission and development. At the SNP and gene level, all subdomains showed overlap, with the exception of RB. However, no overlap was observed at the functional level. In summary, the ADI-R algorithm-derived subdomains related to social communication show a shared genetic etiology in contrast to restricted and repetitive behaviors. The ASD-specific PRS overlapped only partially, suggesting an additional role of specific common variation in shaping the phenotypic expression of ASD subdomains.

The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics.

The genetic architecture underlying Autism spectrum disorder (ASD) has been suggested to differ between individuals with lower (IQ ≤ 70; LIQ) and higher intellectual abilities (IQ > 70; HIQ). Among the identified pathomechanisms, the glutamatergic signalling pathway is of specific interest in ASD. We investigated 187 common functional variants of this neurotransmitter system for association with ASD and with symptom severity in two independent samples, a German (German-ALL: N  = 583 families) and the Autism Genome Project cohort (AGP-ALL: N  = 2001 families), split into HIQ, and LIQ subgroups. We did not identify any association withstanding correction for multiple testing. However, we report a replicated nominal significant under-transmission (OR < 0.79, p  < 0.04) of the AKAP13 rs745191-T allele in both LIQ cohorts, but not in the much larger HIQ cohorts. At the phenotypic level, we nominally replicated associations of CAMK2A -rs2241694 with non-verbal communication in both combined LIQ and HIQ ASD cohorts. Variants PLD1 -rs2124147 and ADCY1 -rs2461127 were nominally associated with impaired non-verbal abilities and AKAP2 -rs3739456 with repetitive behaviour in both LIQ cohorts. All four LIQ-associated genes are involved in G-protein coupled signal transduction, a downstream pathway of metabotropic glutamate receptor activation. We conclude that functional common variants of glutamatergic genes do not have a strong impact on ASD, but seem to moderately affect ASD risk and phenotypic expression. Since most of our nominally replicated hits were identified in the LIQ cohort, further investigation of the glutamatergic system in this subpopulation might be warranted.

The genetic architecture of Autism Spectrum Disorders (ASD) is complex. Common genetic variation has especially been related to high-functioning ASD. In addition, some studies favoured analysis of strictly diagnosed autism individuals, which resulted in more robust findings than the combined analysis of all spectrum individuals. Functional variants modulating EIF4E expression have previously been indicated as risk factors for ASD. Pharmacological modulation of glutamate receptors which regulate EIF4E activity resulted in reduced repetitive behaviours in human and animal studies. Based on these findings, we tested common EIF4E variants for association with overall ASD, with strict autism and with the strict high-functioning autism (strict HFA) subgroup, and their effect on repetitive and/or stereotypic behaviour. We observed over-transmission of rs13109000G in the strict HFA and the strict autism cohort but not in the larger ASD cohort. We report protective effects for the minor allele of rs4699369T on stereotyped and ritualized behaviour in the overall ASD cohort, the strict autism but not in the strict HFA group. In addition, a protective role for rs4699369T and a risk effect of rs12498533G on hand and finger mannerisms was observed. These results need to be replicated in larger ASD and strict autism samples. The predicted impact on transcription through the ASD associated EIF4E variants rs4699369T and rs12498533G as well as the association of the EIF4E interaction partners FMRP and CYFIP1 with ASD point to an mRNA mediated pathomechanism for ASD.

The 16p11.2 gene QPRT, encoding a key enzyme of the kynurenine pathway, has been linked to neurodevelopmental disorders including autism spectrum disorder (ASD). To investigate its role in early human brain development, we inhibited QPRT in stem cell-derived cerebral organoids. QPRT inhibition resulted in reduced organoid size, driven by premature neural differentiation resulting in depleted progenitor populations. Single-cell transcriptomics revealed an excitation/inhibition imbalance, with reduced excitatory and increased inhibitory neuron populations. We observed metabolic stress signatures, including pseudo-hypoxia, oxidative stress, and mitochondrial dysfunction, likely linked to NAD+ depletion and QUIN accumulation following QPRT inhibition. Notably, downregulation of LHX2 and PRDX1 may underlie impaired neural patterning and excitotoxic vulnerability. In addition, we report astrocytic and radial glia dysfunctions, indicating broad effects across multiple cell types. Disease gene enrichment analyses showed significant overlap with ASD-associated genes, especially during early differentiation. These findings suggest the loss or reduction of QPRT to shift neural development and neuronal homeostasis towards an imbalance in excitatory and inhibitory neuronal populations, a mechanism previously associated with neurodevelopmental disorders.